This present invention relates to the synthesis and evaluation of cyclohexyl chelating agents where the complexation of radiometals occurs on one side of the cyclohexane ring and the other side of the ring contains a moiety which allows the chelating agent to be attached to proteins such as antibodies. By the proper choice of radiometal and antibody these antibody-metal conjugates can be used for diagnostic imaging or therapy.
The use of radiometals to label proteins and antibodies provides a wealth of choices of half-life and emissions for various applications (diagnosis and therapy). In addition, radiometal labeling avoids the deleterious oxidation effects experienced in direct iodination reactions. Labeling with metals can also overcome problems of in-vivo deiodination by tumor and normal tissues, particularly when using rapidly internalized antibodies.
Radiometals can generally be attached to antibodies by the use of a "bifunctional chelate" which is first covalently attached to the antibody to form an antibody-chelate conjugate and then bound to the radiometal. The early work in this field used diethylenetriaminepentaacetic acid (DTPA) and its derivatives. This compound has a backbone consisting of three nitrogens separated by two ethylene bridges. The two terminal amine groups each contain two carboxymethyl groups while the internal amine contains one carboxymethyl group. DTPA is generally conjugated to antibodies via its bicyclic anhydride (DTPADA) which forms a covalent amide bond between an antibody amine and one of the carboxylic acid groups of DTPA [Hnatowich, et al. Science 220, 613 (1983)]. This method while convenient has drawbacks. For example, with indium-111 this procedure yields high liver retention and slow body clearance [Goodwin, J. Nucl. Med. 28, 1358 (1987)], as well as a substantial amount of crosslinked antibody (two antibodies linked together by a DTPA bridge). This crosslinking can reduce the immunoreactivity of the antibody, increase liver retention, and decrease tumor uptake.
The introduction of benzyl groups onto the backbone of EDTA and DTPA has been shown to increase the serum stability of .sup.111 In-benzyl EDTA and -benzyl DTPA chelates and antibody conjugates and to decrease the retention of indium in the liver of nude tumor mice [Cole, et al. Nucl. Med. Biol. 13, 363 (1980)]. Unfortunately, this reduction in the retention of indium in the liver has not been observed in patients using these chelating agents [Hnatowich, Seminars in Nucl. Med. 20, 80 (1990)]. With yttrium-88 benzyl-DTPA conjugates were more stable thereby giving higher tumor uptake and lower bone retention than the benzyl-EDTA or cyclic DTPA conjugates [Roselli, et al. J. Nucl. Med. 24, 932 (1989)]in nude tumor mice. The above benzyl-EDTA and benzyl-DTPA ligands where attached to antibodies via 4-isothiocyanato benzyl or a 4-bromoacetamido-benzyl group thereby freeing all five carboxylates for metal complexation. Chelating agents which contain a cyclic backbone are also useful for attaching radiometals to antibodies. For example, the use of macrocyclic polyaminocarboxylates such as the bromoacetamido derivatives of 6-(p-aminobenzyl)- 1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid [Moi et al., Anal. Biochem 148, 249 (1985)]or 2-(p-aminobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid [Moi et al., J. Am. Chem. Soc. 110, 6266 (1988)]have both produced stable copper conjugates while the latter produced stable yttrium conjugates [Deshpande et al., J. Nucl. Med. 31, 473 (1990). The isothiocyanato derivative of the latter compound prepared by a different route has produced stable bismuth conjugates [Gansow and Kumar, U.S. Pat. No. 4,923,985 (1990)].
Trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) is a strong general purpose chelating agents for complexing metals. Its thermodynamic stability constants are generally 1-3 orders of magnitude greater than ethylenediamine tetraacetic acid (EDTA) [Martell and Smith, Critical Stability Constants, Vol. 1., Plenum Press, NY, 1974]. CDTA has been used to remove heavy metals from the body [Kroll et al., Nature 180, 919 (1957)]. To attach CDTA to proteins such as antibodies, CDTA has been converted to its monoanhydride (CDTAMA) [Mease and Svivastava, U.S. Pat. No. 5,021,571 (1991)]. Since CDTAMA is monofunctionalized it does not produce any crosslinked antibodies. .sup.111 In, .sup.57 Co, and .sup.97 Ru labeled immunoconjugates prepared using CDTAMA have shown promising biodistributions in mice. Higher denticity chelating agents containing the cyclohexane ring such as N,N'-(2-aminoethyl)-trans-1,2-diaminocyclohexane-N,N',N",N",N'",N'"-hexaac etic acid (CTTHA) have been prepared and conjugated to antibodies by converting two of the acid groups to activated N-hydroxy succinimide esters [Mease et al. U.S. Pat. No. 5,089,663 (1992)]. In the above cyclohexyl chelating agents, all attachments to the antibody have been through one of the carboxylate groups. This eliminates one coordination group from the ligand and this loss may become critical when trying to complex metals which prefer high coordination numbers. The disclosure of Johnson D.K. (European Patent Application 88101776.8 (1988)) presents a method of attaching a p-nitrobenzyl group to the carbon alpha to one of the carboxyl groups of CDTA to produce N-(carboxymethyl)-N-(trans-2-(bis(carboxymethyl)amino)cyclohexyl))(4-nitro phenyl)alanine. Although not presented in that European application, for this particular compound the ##STR1##
4-nitro group can be reduced to a primary amine and converted to an isothiocyanato group for immunoconjugation. While a reasonable method, this approach is only general for the production of polyaminocarboxylate ligands and the juxtaposition of the benzyl cyclohexyl groups may sterically inhibit metal complexation.
In order to move the conjugating moiety to the other side of the cyclohexane ring, Stavrianopoulos (U.S. Pat. No. 4,707,440 (1987)) has prepared the bromohydrin compound 1 ##STR2## 1.times.=-Br 2.times.=-SCH.sub.2 CH.sub.2 C(O)NHNH.sub.2
3.times.=-SC.sub.6 H.sub.6 NH.sub.2
This was converted to 5-hydroxy-CDTA-4-B-thioproprionic acid hydrazide 2. This was conjugated to BSA using 1M HCl and 1M NaNO.sub.2. Also the 4-aminophenyl derivative 3 was prepared and conjugated to BSA by the formation of a very reactive diazonium salt. While demonstrating the ability of attaching CDTA to a protein on the back side of CDTA the methods described are not convenient and may not be applicable to sensitive antibodies.
4-amino CDTA is describe briefly in the disclosure of Engelhardt et al. (European Patent Application #106112.2). No details of this preparation which starts with 1-hydroxy-3,4-dinitro benzene are given. Also no stereochemistry is given on the reduction product 4-hydroxy-1,2-diaminocyclohexane or subsequent intermediates including 4-amino CDTA. The diamines must be in the trans-diequatorial position for effective metal complexation.